determination of nitrate and nitrite content in several vegetables

An-Najah National University Faculty of Graduate Studies

Determination of Nitrate and Nitrite Content in Several Vegetables in Tulkarm District.

By Afaf Ghaleb Hafiz Abu-Dayeh

Supervisor Dr.Hassan Abu Qaoud

Co-Supervisor Dr.Nidal Zatar

Submitted in Partial Fulfillment of the Requirement for the Degree of Master in Environmental Sciences, Faculty of Graduate Studies, at An-Najah National University, Nablus, Palestine.

July,2006

An-Najah National University

Faculty of Graduate Studies

Determination of Nitrate and Nitrite Content in Several Vegetables in Tulkarm District.

By Afaf Ghaleb Hafiz Abu-Dayeh

Supervisor Dr.Hassan Abu Qaoud

Co-Supervisor Dr.Nidal Zatar

Determination of Nitrate and Nitrite Content

in Several Vegetables in Tulkarm District.

By

Afaf Ghaleb Hafiz Abu-Dayeh

This Thesis was defended successfully on ……………. and approved by :

Committee Members Signature

1- Dr.Hassan Abu Qaoud…… ……………………..

2- Dr.Nidal A. Zatar………… ………………………

3- Dr.Amal Hudhud………… ……………………….

4- Dr. Raqi Shubita………… ………………………..

II

Dedication

My grateful thanks and appreciation are for my precious parents, for their

encouragement, effort, and patience. My grateful thanks are also for my

sisters, and for my husband who was very supportive and guided me all the

way, and for everything they all did for me until I finished this work. My

thanks are also for all my friends for their support.

III

Acknowledgment

Grateful acknowledgment is made to Dr.Hassan Abu Qaoud and Dr.Nidal

Zatar for their full and continuous supervision of my work.

Great thanks are extended to the staff working in the chemical, biological,

and drugs analysis center for their valuable assistance during my work on

the crops analysis, and for everyone helped me or support me directly or

indirectly to complete this work.

IV

Table of contents

Branch Content Page

Dedication. I

Acknowledgment. II

List of Contents. III

List of Tables. IV

Abstract. V

1. Chapter One: Introduction

1 Introduction. 2

1.1 Nitrate and Nitrite Effect. 7

1.2 Study Area. 11

1.3 Objectives. 17

2. Chapter Two: Literature Review

2.1 Nitrate and Nitrite in Drinking Water. 20

2.2 Nitrate and Nitrite in Animals. 20

V

2.3 Nitrate and Nitrite in Vegetables. 21

3. Chapter Three: Materials and Methods.

3.1 Materials and Methods. 35

3.2 Instruments. 35

3.3 Sample Collection. 36

3.4 Preparation of Reagents and Standard Solutions. 38

3.5 Nitrate and Nitrite Extraction. 38

3.6 Quantitative Determination. 39

3.6.1 Quantitative Determination of Nitrate. 39

3.6.2 Quantitative Determination of Nitrite. 39

3.7 Statistical Analysis. 40

4 Chapter Four: Results. 42

5 Chapter Five: Discussion. 52

6 Chapter Six: Conclusions. 61

7 References. 63

VI

8 Appendices. 70

9 Abstract in Arabic. 80

VII

List of Tables:

Table Address Page

Table (1) Nitrate intakes from sources other than food additives

in the middle and far eastern.

5

Table (2) Nitrite intakes from sources other than food additives

in the middle and far eastern.

6

Table (3) The land use classification in Tulkarm district. 12

Table (4) The average climatic parameters for Tulkarm district

for the year 2005.

14

Table (5) The area, production, and value of different crops in

Tulkarm district.

15

Table (6) Number of samples collected from the different

locations for each vegetable type.

37

Table (7) Average nitrate (NO3) content in the vegetables

surveyed in Tulkarm district.

43

Table (8) Average nitrite (NO2) content in the vegetables

surveyed in Tulkarm district.

44

VIII

Table (9) Average nitrate (NO3) content in large size cucumber

fruits.

46

Table (10) Average nitrate (NO3) content in small size cucumber

fruits.

46

Table (11) Average nitrite (NO2) content in large size cucumber

fruits.

47

Table(12) Average nitrite (NO2) content in small size cucumber

fruits.

47

Table (13) Average nitrate concentration (NO3) in (mg/Kg) in

the essence of cucumber fruits.

49

Table (14) Average nitrate concentration (NO3) in (mg/Kg) in

the peel of cucumber fruits.

49

Table (15) Average nitrite concentration (NO2) in (mg/kg) in the

essence of cucumber fruits.

50

Table (16) Average nitrite concentration (NO2) in (mg/kg) in the

peel of cucumber fruits.

51

Table (17) Comparison of nitrate (NO3) content in the vegetables

surveyed in Tulkarm district, and the nitrate content

54

IX

reported in other studies.

Table (18) Comparison of nitrite (NO2) content in vegetables

surveyed in Tulkarm district, and the nitrite content

reported in other studies.

55

X

Appendices:

Appendix Address Page

Appendix (a) The area (dunum) cultivated with vegetable

croups in Tulkarm district in (2003-2004).

70

Appendix (b) The area (dunum) cultivated with unprotected

vegetables and field crops in Tulkarm district

(2003-2004).

72

Appendix (c) The most cultivated trees and their areas (dunum)

in Tulkarm district for the year (2003-2004).

74

Appendix (d) Descriptive statistics of chemical and physical

parameters of the wells studied in Tulkarm

district.

76

Appendix (e) Physical and chemical results obtained from the

wells studied in Tulkarm district.

77

A map for Tulkarm district/governate 79

XI

Determination of Nitrate and Nitrite Content

in Several Vegetables in Tulkarm District. By

Afaf Ghaleb Hafiz Abu-Dayeh Supervisor

Dr.Hassan Abu Qaoud Co-Supervisor Dr.Nidal Zatar

Abstract

A study of the content of nitrate and nitrite in few different vegetables in

Tulkarm district was conducted. This district is part of the West Bank land

(Palestine). A total of 75 samples of five different types of vegetables

(tomato, cucumber, onion, potato, and cabbage) were collected from

randomly selected farms in three different areas in Tulkarm district

(Thenabe, Al-Sha'raweya, and Al-Kafriyat). The nitrate content in the

samples was analyzed using HANNA spectrophotometer (HI93728-0),

while the nitrite content was analyzed using the AOAC Official Method

973.31.

The results showed that the type of the vegetable has a great effect on the

nitrate content, the highest nitrate content was found in potato with an

average of 253.13mg/Kg, while the lowest nitrate content was found in

tomato with an average of 16.95mg/Kg. The nitrate content was affected

by fruit size; the small cucumber size shows a higher nitrate content than

XII

the large size fruits; It was found that the peels of the cucumber fruits

contain higher nitrate content than the essence. Cabbage as a leafy

vegetable shows a high nitrate content. The nitrite concentrations were low

(less than 1mg/Kg) in all test crops. These levels of nitrite are considered

acceptable. The different locations show no significant effect on the nitrate

and nitrite contents in vegetables tested in this study. This study high lights

an important issue which was not investigated before. Under the conditions

of the study, the results indicated acceptable levels of both nitrate and

nitrite content. However more work is needed to elaborate these results

with more samples and different crops.

1

Chapter One

Introduction

2

Introduction

Pollution issue is now among the most important issues, that the

world countries are interested in, since this issue has a great effect on all

life aspects and causes great threaten to animals, plants, environment, and

human health. The pollution problems rose up after the industrial

evolution.

Nitrates and nitrites seemed to be among the chemicals that may

cause pollution; many studies have expected the effect of these compounds

on the environment and on the living health. These studies focused on the

nitrate and nitrite contents in water sources and in vegetables consumed by

humans. In order to control the nitrate and nitrite intake by consumers in

general and on babies in particular who are the most vulnerable to the

adverse effects of these two compounds, a maximum acceptable limit of

these compounds were suggested.

The Acceptable Daily Intake (ADI) of nitrate and nitrite set by

European Commission's Scientific Committee for Food (ECSCF), is

3.7mg/Kg body weight, and 0.06 mg/Kg body weight, respectively

[WHO,1995] [18, 42].

3

For methamoglobinaemia in infants, it was confirmed that the

existing guideline value for nitrate ion in drinking water of 50 mg/L is

protective. For nitrite, human data reviewed by (JECFA) support the

current provisional guideline value of 3 mg/L [15].

Agriculture is considered the major source of nitrate and nitrite in

the environment [16]. Fertilizers use is rapidly increased, causing

excessive nitrogen loading to the environment [16, 47]. Globally, the use

of fertilizers was increased from less than 14 million tones in 1950 to 135

million tones in 1996 [67, 69 ]. In 1998, the world used 137 million metric

tons of chemical fertilizers. [24]

In our areas (in Palestine), farmers use higher amounts of fertilizers

than they should, either due to their ignorance, or because they want to

increase their production quickly when the prices of these products in

markets are high. Plants can not utilize all the added fertilizers, so the

excessive amounts will be leached out by irrigation water through the soil

to reach ground water, or it may dissolve in run off water and flows into

streams or lakes and rivers [10]. Nitrogen compounds are also accumulates

in some plant tissues [10].

More than three quarter of our average nitrate intake comes from

vegetables [54], which provide about 80% of the average daily dietary

intake [15]. Vegetables that may accumulate nitrate in their tissues are

4

leafy vegetables such as spinach, lettuce, and cabbage, or root crops like

carrot, beetroots, potatoes, radish, and others like cauliflower, beans, and

peas. The amount of nitrate in vegetables like lettuce is usually ranges

between 90 and 3520 ppm [54].

The intakes of nitrate and nitrite from food were calculated as a

global level on the basis of mean food consumption in the GEMS/Food

regional diets [WHO, 1998], and the mean concentrations in food in

Europe from the submitted data. Intake from drinking water was added,

assuming a water consumption of 2 L/day. The mean concentration in

water that was used in the intake calculations was 4 mg/L for nitrate and

0.3 mg/L for nitrite (Table 1&2), which are representative of the usual

concentrations found in water [WHO, 1998]. An average body weight of

60 Kg was used for the global intake assessment [27].

5

Table (1): Nitrate intakes from sources other than food additives in the middle and far eastern [26].

Food Nitrate

mg/Kg

Diet

Middle Eastern Far Eastern

Consumption (g/day)

Intake (mg/day)

%total intake

Consumption (g/day)

Intake (mg/day)

%total intake

lettuce 1700 2.3 4.0 10 0 0 0

spinach 1900 .05 .093 2 0 0 0

Chinese cabbage

3500 0.1 0.35 1 0.1 0.35 1

onion 110 23 2.5 6 12 1.3 5

potato 180 5.9 1.1 3 19 3.4 13

cabbage

340 5 1.7 4 9.7 3.3 12

tomato 15 82 1.2 3 7 0.1 0

Fruit* 25 19 0.48 1 78 1.9 7

water 4 2000 8 20 2000 8 29

ADI%* 20 10

* Fruit: total fruit, minus apple and pear.

* ADI: Acceptable Daily Intake

6

Table (2): Nitrite intakes from sources other than food additives in the middle and far eastern [26].

Food Nitrite (mg/Kg)

Diet

Middle Eastern Far Eastern

Consumption (g/day)

Intake (mg/day)

%total intake

Consumption (g/day)

Intake (mg/day)

%total intake

Potato 0.7 5.9 0.0041 0 19 0.013 1

vegetable

0.6 230 0.14 7 180 0.11 5

Cereals 2.6 430 1.1 54 450 1.2 59

Fruit* 0.5 200 0.1 5 85 0.043 2

ADI%* 50 50

* Fruit: total fruit.

* ADI: Acceptable Daily Intake.

7

1.1. Nitrate and nitrite effect:

The high concentration of nitrate has adverse effects on environment,

animals and humans:

a. Environment: the high concentrations of nitrate in water causes a

phenomenon known as "Eutrophication", which means an excessive

growth of the algae in water which consumes the oxygen gas dissolved

in water causing the death of fishes in that water [36].

b. Animals: especially ruminant animals such as cows, sheep and goats

[5]. When ruminants consume feed with high nitrate levels, the nitrate

can be converted to nitrite, which causes both nitrate and nitrite

accumulation in the rumen [5].

As a result of the accumulation of both nitrate and nitrite in animal

rumen, it causes acute and chronic symptoms which run as reduction in

weigh gain, reduction in milk production, low appetite, aborted

breathing, blue coloring of mucus membrane, rapid heartbeat,

abdominal pain, vomiting, reproductive problems, abortions, and

premature death of calves [5, 63].

The acute nitrate poisoning causes death, because the nitrate is

reduced to nitrite in the rumen by bacteria. Nitrite is highly toxic

8

because it combines with hemoglobin and form methemoglobin which

is enabling to carry oxygen [3, 5, 33, 63].

A death occurs within few hours after the ingestion of a high nitrate

feed [5].

c. Human: Nitrate itself is not toxic; however, the conversion of nitrate

to nitrite in human and animal bodies is very dangerous if it

accumulates in high concentrations [5].

On the other hand, the following could be occurring as a result of the

accumulation (or uptake) of large dose of nitrate for human health:

1. Methemoglobenemia [62] [15, 68]: drinking water and vegetables

are the major sources of nitrate consumed by human stomach [67].

Nitrate is reduced to nitrites which combine with hemoglobin to

form methemoglobin (metHP). Methemoglobin is a compound that

can not combine with oxygen, and that decreases the capacity of the

blood to transport oxygen from lungs to body tissues causing a

condition known as “Blue Baby Syndromes” or

“methemoglobenemia” [47].

NO2 + oxyHb (Fe2+)---- metHb (Fe3+) + NO3 [15]

9

The normal met HP level in humans is less than 3% in infants under

three months of age. However hypotoxic signs may develop at about

20%, while death occurs at 50% metHP or higher [15, 66].

Because of their high stomach acidity, infants less than one year old

(3-6 months old), are highly infected by the methemoglobenemia, this

acidity increases the conversion of nitrate to nitrite by providing an

appropriate environment for the nitrate reducing bacteria [WHO,1996]

[16].

Other groups that may be risk to form met HP are pregnant women

[15, 22, 63], which may cause birth defects [62, 63] and miscarriages

[63]. Also people who have deficiency in glucose-6- phosphate

dehydrogenase or metHb reductase may be at risk to form metHP [15,

22].

Adults with lower gastric acidity can be infected with metHP.

Fatalities have been reported after single intake of 4-50 g of nitrate

(equivalent to 67-833 mg of nitrate per Kg of body weight)

[WHO.1996].

The symptoms related to high levels of metHP in blood includes

bluish discolorate of skin, headache, dizziness difficulty in breathing, in

sever cases damage to brain and death may occur.

10

2. Carcinogenicity: nitrite reacts in stomach with nitrosatable

compounds to form N-nitraso compounds [36] [15, 16]. These

compounds have been found to be carcinogenic [10] [15, 22, 33].

The US National Research Council found an association between

high nitrate intake and gastric and esophageal cancer [63].

High levels of nitrate intake were also linked with the Non-

Hodgkin’s lymphoma [63], bolder cancer [63], pancreatic cancer [14]

and stomach cancer [1] [25, 54].

High levels of nitrate were also linked with the infection with the

diabetes, the occurring of some birth defects, and miscarriages [63].

11

1.2. Study area:

The study was conducted in Tulkarm district as an agricultural area.

Tulkarm district lies on the western north part of West Bank in Palestine. It

is bounded by Nablus and Jenin districts in the North, West, and South,

and by the 1948 cease-fire line in the East.

Tulkarm district covers approximately 334,530 dunum.

Table (3) shows the classification of the land use in Tulkarm district,

which shows inefficiency in the use of land, where both Palestinian built-

up and Israeli settlements are built on lands of extremely rich soil for

cultivation. This has led to the spread of built-up areas and industries at the

cost of agricultural land.

12

Table (3): The land use classification in Tulkarm district [74].

Land use Area/ dunum % of land

Palestinian Build up Areas

18020.5 5.39

Israeli Settlements 3170 0.95

Israeli Military Bases 70.5 0.02

Industrial Park 550 0.16

Nature Reserves 1730.5 0.52

Forests 4140 1.24

Cultivated Areas 128100.5 38.3

Others* 178720.4 53.42

Total 334530 100

* Unused land or land used for grazing.

- The climate: Climate of the study area is a Mediterranean type with

hot summers and warm winters.

- Precipitation: the rainy season starts in October and continues

through May. Almost 70% of the annual rainfall occurs between

December and February, while 20% of the annual rainfall occurs in

October and November. The mean annual rainfall in Tulkarm city is

614.7 mm for the period from 1952 to 1995 (Tulkarm Agricultural

Department).

13

- Humidity: humidity in Tulkarm district reaches high values with an

annual average of 69.6%, which rises in winter to 75.9%in February,

while it reaches its lowest value of 62.4% in May.

- Temperature: in Tulkarm district, the mean annual maximum

temperature is 22.3Co, and the mean annual minimum temperature is

15.6Co.

- Winds: the wind direction in Tulkarm district mainly lies between

the southwest and northwest with mean annual wind speed of

3.4Km/hr. Khamaseen winds blow over the area in the spring full

with sand and dust, causing rising temperature and drop in humidity.

The main daily wind speed from April to June is 3.2Km/hr.

14

Table (4): The average climatic parameters for Tulkarm district for the year 2005.

Month Max. Temp(Co)

Min.

Temp(Co)

Average

Temp(Co)

Humidity

(%) Wind speed

(Km/hr)

Rainfall

(mm) (1961-1995)

Jan. 19.0 9.2 15.5 64 4.0 218.8

Feb. 18 8.0 15.0 71 4.0 139.9

Mar. 22.5 10.5 18.0 63 3.0 17.1

Apr. 26.0 13.1 22.5 52 4.0 4.8

May 28.0 16.0 24.0 58 4.0 _

June 31.0 21.0 28.0 58 4.0 _

July 33.0 24.0 30.0 60 4.0 _

Aug 33.0 25.0 30.0 60 3.0 _

Sep. 34.3 23.1 30.0 59 4.0 _

Oct. 29.0 20.0 29.0 53 2.0 7.7

Nov. 23.7 11.7 20.6 59 5.0 56.6

Dec. 21.0 11.9 17.5 69 2.0 140.9

Average

22.3 15.6 18.95 69.6 3.4

Total 663.3

*Tulkarm Weather Station.

15

Tulkarm is a very fertile area which explains its relative high

population in comparison to other districts 583 person/km2, while the

population in Ramallah, Hebron, and Beithlahem are 200, 245, and 230

person/km2, respectively.

The total area of Tulkarm is about 253,950 dunum. The arable area

is 209,064 dunum, but the actually cultivated area is 138,368 dunum

(ministry of agriculture 2004/2005). These areas are mainly cultivated with

fruit trees, vegetables, and field crops.

Table (5) shows that fruit trees –mainly un-irrigated olive and

irrigated citrus- forms the greatest area and it worth 27,655,000 U.S.A$,

next comes the field crops and then the vegetables.

Table (5): The area, production, and value of different crops in Tulkarm

district.

Crop Area/dunum Production/ton Worth (1000 dollar)

Fruit trees 134,264 39,542 27,655

Vegetables 10,302 53,519 22,025

Field crops 482,848 6170 2,765

*Ministry of agriculture in Tulkarm.

16

In general, planting the open, protected and irrigated vegetables is

considered the most widely spread type of agriculture; that is the number

of green houses is 6642 and the number of high tunnels is 1375.

For the protected crops, the cucumber forms the most planted

vegetables, with an area of about 1500 dunum with an average production

of 6-8 tons/dunum.

Next to cucumber comes the tomato and other vegetable crops like

beans and pepper. The faba bean, chick-pea and Egyptian cucumber are the

most un-irrigated vegetable crops in Tulkarm district. For the irrigated

field crops; the most planted crops are wheat and barely which occupies

6894 and 1339 dunum, respectively.

For the fruitful trees, citrus trees –mostly orange trees- are the most

planted irrigated trees which occupy 3700 dunum.

Finally, the olive trees are the mostly planted un-irrigated trees, and

it occupies 114,785 dunum according to Tulkarm Agricultural Department

for the year 2003/2004. [9, 44]

17

1.3. Objectives:

Pollution issue has become one of the most important public

awareness issue. The excessive use of the pesticides and fertilizers in

agriculture with the threat of these chemicals in crops and water. Therefore

great wonders have been raised about the cleanness and the quality of the

vegetable crops grown in West Bank areas.

This work is a trial to find out the status of some chemical residues in

vegetables. Therefore the aim of this work is to examine some of the most

common vegetables grown in Tulkarm area for its nitrate and nitrite

contents.

The objectives of the present work:

1- Determine the level of nitrate and nitrite in tomato, cucumber,

potato, onion, and cabbage crops planted in Tulkarm district.

2- Determine the effect of size of fruits on the level of nitrate and

nitrite

3- Comparison of nitrate and nitrite level in peel and essence of crops.

18

4- Comparing the content of nitrate and nitrite between underground

crops (potato and onion) and aboveground crops (tomato and

cucumber).

5- Comparing the nitrate and nitrite content between the onion crop and

potato crop.

6- Comparing the content of nitrate and nitrite between the leafy crops

(cabbage), and other edible crops.

19

CHAPTER TWO

LITERATURE REVIEW

20

Chapter Two

Literature Review:

2.1. Nitrate and nitrite in drinking water.

In a study surveyed by [32], the association between nitrate and

atrazine from drinking water, and stomach cancer was studied. Existing

data on the incidence on spectic types of cancers, contamination of

drinking water with atrazine and nitrate, and related agriculture practices

for the 40 ecodestricts in the province of Ontario, were obtained. It was

found that the atrazine contamination levels range 50-649 ng/L, Maximum

Acceptable Concentration (MAC) was 60 mg/L, were positively associated

with stomach cancer incidence and negatively with colon cancer incidence.

Nitrate levels (range 0-91 mg/L, MAC = 10 mg/L) were negatively

associated with stomach cancer incidence.

2.2. Nitrate and nitrite in animals.

Hungry cattle and sheep introduced to stockyards containing a

dominant growth of Dactylodenium radulans (button grass), suffered from

acute nitrate-nitrite toxicity in four incidents in Island Queensland between

1993 and 2001. Deaths ranged from 16 to 44%. In all accidents,

21

methemoglobenemia was noted at necropsies. An aqueous humor sample

from one dead steer contained 75 mg nitrate/L, and from one dead sheep

contained 100 mg nitrate/L, and 50 mg nitrite/L (normal = 5 mg nitrate/L).

The dry button grass was toxic. The nitrate content of button grass from

within the stockyards ranged from 4% to 12.9% as potassium nitrate

equivalent in dry matter [40].

2.3. Nitrate and nitrite in vegetables.

Nitrate content was determined in several edible vegetables in Italy

in 1996-2002. It was found that the highest content of nitrate was detected

in chicory (6250 mg/kg) and rocket (6120 mg/kg), which are consumed in

large quantities in some regions of Italy. Green salad and lettuce contained

less nitrate (highest values were 4200 mg/Kg and 3300 mg/kg,

respectively), but because they are consumed more generally, they

provided 60% of the total intake of nitrates. Only a few samples were

above the legal limits, with seasonal variation. Significantly higher nitrate

content was found in organically grown green salad and rocket than in

those conventionally produced. These data indicates that the average intake

of nitrate from leafy vegetables is below the acceptable daily intake, i.e.

3.7 mg nitrate ion/Kg body weight per day, but the total intake should be

monitored to protect groups at risk, such as children and vegetarians [39].

22

Nitrate (NO3) and nitrite (NO2) contents of various vegetables

(Chinese cabbage, radish, lettuce, spinach, soybean sprouts, onion,

pumpkin, green onion, cucumber, potato, carrot, garlic, green pepper,

cabbage and Allium tuberosum Roth known as Crown daisy) were

reported by (Chung et al., 2003). Six hundred samples of 15 vegetables

cultivated during different seasons were analyzed for nitrate and nitrite by

ion chromatography and ultraviolet spectrophotometry, respectively. No

significant variance in nitrate levels was found for most vegetables

cultivated during the summer and winter harvests. The mean nitrates level

was higher in A.tuberosum Roth (5150 mg/kg) and spinach (4259 mg/kg),

intermediate in radish (1878 mg/kg) and Chinese cabbage (1740 mg/kg),

and lower in onion (23 mg/kg), soybean sprouts (56 mg/kg) and green

pepper (76 mg/kg) compared with those in other vegetables. The average

nitrite contents in various vegetables were about 0.6 mg/kg, and the values

were not significantly different among most vegetables. It was observed

that nitrate contents in vegetables varied depending on the type of

vegetable and were similar to those in vegetables grown in other countries.

In Nigeria the contents of nitrate, nitrite, and ascorbic acid were

determined in four samples of commercial and fifteen samples of home –

prepared complementary infant foods. For the commercial food samples,

the nitrate and nitrite values ranged from 3.1-3.9 mg NO3-N/100 g and 5.0-

23

16.0 ug NO2-N/100 g, respectively, while the ascorbic acid content ranged

from 6.0-13 mg/100 g. For the home prepared complementary infant foods,

the nitrate and nitrite content varied according to recipes with maximum

values found in foods containing vegetables and legumes. The highest

nitrate levels were found in yam and vegetables pottage (25.1 mg NO3-

N/100 g), and that of nitrite in pureed spinach vegetable (72.0 mg NO2-

N/100 g). The ascorbic acid content was very low. However, the nitrate

and nitrite levels of these complementary foods were below tolerance

levels, and these foods do not pose any health problem for infants [62].

In a study comparing the nitrate reductase activity (NRA) in

different tissues of poplar plant, it was found that NRA was greater in

leaves, and the highest leaf NRA was found in young leaves. Leaf and root

NRA increased with increasing nitrate supply, whereas stem NRA

remained constant. Leaf NRA was at least 10-fold greater than root NRA

at all external nitrate concentrations. Nitrate reductase abundance increased

in all tissues with increasing nitrate availability, and nitrate reductase

abundance was at least 10-fold greater in leaves than in stems or roots at

all nitrate availabilities. Tissue nitrate concentration increased with

increasing nitrate supply and was greater in roots than in stems and leaves.

Photoperiod influenced NRA, with leaf NRA declining in nitrate-fertilized

plants with short daily photoperiods (8-h). It was concluded that different

24

tissues of poplar vary in nitrate assimilation with little nitrate assimilation

occurring in roots and the most nitrate assimilation taking place in leaves

[12].

The influence of nitrogen concentration and the ratio of NO3 to NH4

on the distribution of inorganic elements within the tissues of cucumber

fruit grown on rockwool were studied. Nitrogen was supplied at three

nitrogen concentrations and four ratios of NO3 to NH4. Increases in the

total nitrogen concentration within the nutrient medium significantly

increased the NO3 content of all the fruit tissues, but this effect was most

pronounced in the skin, neck and apical region. The concentration of K,

Ca, Mg and NO3 in all regions of the fruit was higher when NO3

constituted 75% or more of the total nitrogen in the nutrient medium, but

was reduced by increasing concentrations of NH4. The Mn content of the

tissue in the central region of the fruit was reduced by NH4 ions, whereas

the Cu content of this tissue increased. No significant effect of nitrogen

form on the B, Fe, Zn content of fruits was detected. In contrast, the B

content of the apical tissue and the Zn content of the skin declined

significantly with increasing nitrogen level. The highest concentrations of

K and NO3 were found in the neck and skin tissues, whereas the highest

concentrations of Mn and Cu were observed only in the fruit skin. Mg

concentrations were lowest in the tissues of the fruit neck, while B

25

concentrations were highest in tissues near to the site of style abscission

[31]

The contents of nitrate and nitrite in potato, cabbage, Chinese

cabbage, scallion (shallot), celery, cucumber, tomato, eggplant and wax

gourd taken from the north China market from 1998 to 1999 were

determined. The highest content of nitrate was found in celery followed by

Chinese cabbage, cabbage, scallion, wax gourd and eggplant. For all the

products, a great variation in the content of nitrate was found. Generally,

the nitrite content was low. The average intake of nitrate and nitrite from

these vegetables was estimated as approximately 422.8 and 0.68 mg/day,

respectively [73].

Nitrate accumulation in 48 varieties of vegetables was carried out at

different seasons in China. The results showed that nitrate-nitrogen

concentrations in 20 vegetables reached Pollution Level 4 (NO3-N > 325

mg/kg), which accounted for 41.7% of the total number of the sampled

vegetables and included all of the leafy, and most of the melon, root, onion

and garlic vegetables. Among them, 5 leafy vegetables even exceeded

level 4 (NO3-N > 700 mg/kg). Although leafy vegetables were usually apt

to heavily accumulate nitrate, most of them were with nitrate-nitrogen

concentrations lower than Level 3 (NO3-N < 325 mg/kg) in leave blades.

26

Further investigation showed that vegetable soils accumulated more

nitrates in each layer from 0 cm to 200 cm than did cereal crop soil. The

total amount of residual nitrate-nitrogen was 1358.8 kg/hm2 in the 200 cm

soil profile of usual vegetable fields, and 1411.8 kg/hm2 and 1520.9

kg/hm2 in the 2-years and the 5-years long plastic greenhouse fields,

respectively. However that in the cereal crop fields was only 245.4 kg/hm2.

Nitrate residual in vegetable soils formed serious threats to underground

water in vegetable growing areas [65].

Many researches have been conducted on this important issue, in the

region; however, no such work has been conducted in our area (Gaza and

West Bank, in Palestine). The following is a summary of the related work.

In a study conducted in Jordan, the nitrate content of several

vegetable produced in the Jordan valley was measured. Results of the

survey indicated that there was a general increase in the nitrate and nitrite

levels in the sampled fruits. Tomatoes had lower levels of nitrate and

nitrites than squash and cucumber. The same vegetables growing in plastic

houses had higher levels of nitrate than those grown in open fields or

tunnels. In addition, large size cucumber and squash fruits and their pulp

had significantly (p<0.01) lower nitrate levels than small size fruits (88 -

98%), and their peels (52 - 79%).

27

The nitrate level ranged between 20.0-160.5 mg/kg in cucumbers,

63.9-248.7 mg/kg in squash, and below detection level in tomatoes. The

levels of nitrate and nitrite found in vegetables surveyed in this study

showed that the results are below the levels reported in similar products

grown elsewhere in the world, especially in European countries [7].

In another study, the effect of different sources of nitrogen, time,

and methods of application on growth and nitrogen content in potato plants

was studied in Kenya. It was found that the late application of nitrogen

fertilizers resulted in higher accumulation in both leaves and potato tuber

[20].

In another study, cucumber plants (Cucumis sativus L. cv.Brunex)

were grown under controlled conditions in an experimental greenhouse and

treated with four rates of nitrogen in the form of KNO3 (N1, 5 g/m2; N2, 10

g/m2; N3, 20 g/m2; and N4, 40 g/m2). The intermediate nitrogen rates (N2

and N3) gave higher utilization of NO3- in the leaves (highest NR

activities) than treatment N1 (inadequate) and N4 (excessive). This latter

rate (N4) appears to result in excessive foliar assimilation of NO3- , thereby

increasing amino acids and proteins and inhibiting or reducing NR activity.

N2 and, especially, N3 treatments strengthened the translocation of organic

nitrogenous compounds (amino acids) towards the fruit, which enhanced

the commercial yield [57].

28

The effects of cultivar and harvest time on the nitrate and nitrite

content of the edible parts of several vegetables were studied. Harvest date

was found to have a significant effect (P=0.05) on the nitrate content of the

open-field-grown spinach, cabbage, and squash, and the nitrite content of

the open-field-grown spinach, lettuce, and cabbage. Late-harvested

vegetables had the lowest nitrate levels, while the pattern of their nitrite

content was irregular with respect to the dates that gave highest nitrite

levels in each vegetable. Harvest date had no significant effect on either

nitrate or nitrite content of the greenhouse-grown vegetables.

Cultivar had a significant effect (P=0.05) only on the nitrate content of the

greenhouse-grown tomatoes and squash, while it had no effect on either

the nitrate or the nitrite content of all other vegetables irrespective of their

cultivation method, although their levels in the greenhouse-grown

vegetables were higher than those grown in open fields. Nitrate levels in

these vegetables were generally low (lowest average of 0.13 mg/100 g in

open-field-grown cauliflower, and highest of 4.77 mg/100 g in greenhouse-

grown squash). Nitrite levels, on the other hand, were similar to those

reported elsewhere in the world, ranging from non-detectable levels in

open-field-grown cauliflower, to a maximum level of 0.43 mg/100 g in

greenhouse-grown squash.

29

A highly significant, although low, positive correlation (r = 0.55, P = 0.01,

n = 108) was found between nitrate and nitrite contents of the greenhouse-

grown vegetables, compared to a non-significant, and much lower

correlation between the two variables in the open-field-grown vegetables

[8].

A research in Lowa community studied the relation between the

nitrate and nitrite levels in drinking water and dietary sources with

pancreatic cancer. The study suggests that long term exposure to drinking

water with low levels of nitrate (about 10 mg/L) is not associated with

pancreatic cancer, while the consumption of dietary nitrate from animal

products may increase the risk [14].

The correlation between consuming food with high nitrate levels and

the formation of carcinogenic nitrosoamines compounds was studied. This

study concluded that the consumption of risk nitrate diet increased the risk

of formation of carcinogenic nitrosoamines [36].

In another study, the nitrate and nitrite levels were determined in

some common vegetables, which eaten row. In many samples the nitrate

concentration was higher than the concentration in spinach (well known as

rich source of nitrates), also commercial lettuce had a very high nitrate

amount, while lettuce cultivated without fertilizing contained low amounts

of nitrates [19].

30

In Poland, a study applied to determine the contents of nitrate and

nitrite in samples of beetroot, white cabbage, parsley root, carrot, and

potato. Mean nitrate concentration in products from conventional

alloments, ranged from 203 in potatoes to 843 in beetroot (mg/Kg wet wt)

vs 145 to 350 (mg/Kg wet wt) in the corresponding ‘ecological’ products.

Mean nitrite concentration (mg/Kg wet wt) in the conventional products

ranged from 0.53 in carrots to 1.54 in beetroot, vs 0.34 to 0.42 in the

corresponding ‘ecological’ products [34].

In another study, the content of nitrate and nitrite in 28 commercial

samples of (a) black tea, and 24 of (b) herbal and medical teas were

determined. Nitrate (KNO3) concentration in (a) ranged from 43.9 to

139.2, and in (b) ranged from 70 to 6207 (mg/Kg).

The effect of shading, harvesting time, culture system, and

concentration of N, P, and Ca on accumulation of nitrate in leaves of

Chinese cabbage were studied. Increasing shading rate increased the nitrate

contents; this phenomenon was greater in autumn than in winter, while leaf

nitrate content was lower when the plant was harvested in the late

afternoon. Nitrate contents increased in the nutrient solution under a non

circulating system, whereas no significant differences were found under a

circulating system. Increased potassium concentration decreased the nitrate

levels in the leaves, while increased concentration of calcium significantly

31

decreased nitrate levels in summer, but not in winter [72].

The effect of time of harvesting and fruit size on the nitrate content

in greenhouse cucumber was studied. The effect of fruit size was

negligible. Harvesting time had a significant influence, the content of

nitrate declined by 51% and 71% for the Sandra and Boloria var.

respectively between the first and last harvesting. Sandra var. had a 62%

higher nitrate content than the Boloria var [52].

In the Province of Lodz, a study was applied to determine the

contents of nitrates and nitrites in 28-35 samples of 10 different vegetables

(anion, cabbage, carrot, cucumber, parsley, tomatoes, leaks, lettuce, celery,

and potatoes). Mean contents of NaNO3 mg/Kg ranged from 41.2 mg/Kg

in tomatoes to 1389 mg/Kg in lettuce, and mean nitrite contents of NaNO3

mg/Kg ranged from 0.11 mg/Kg in tomatoes to 1.47 mg/Kg in lettuce [38].

Another study determined the contents of nitrate and nitrite in 33

samples of (fresh and boiled carrot, parsley, leek, celery and potato) was

determined in another study. The nitrate content were reduced with boiling

by approximately 50% and the nitrite content by 37-71% while the

contents of nitrates and nitrites in carrot juice and in the raw carrot were

the same [37].

32

In a study in Queensland, vegetables were collected and analyzed

for nitrate and nitrite residues. Vegetables were collected near peak

harvest. The survey also included a small sample of hydroponics produce

levels of nitrate in potatoes, cabbages and beets were higher than those

reported in other surveys. The median NO3-N concentration measured in

hydroponics lettuce (465 mg/kg NO3 -N) was twice more than the median

cancer far field grown lettuce [35].

The content of KNO3 and NaNO2 in samples of fresh vegetables,

frozen vegetables, apple, soft fruits, frozen fruits and James was

determined. Some vegetables contained >1000 mg KNO3/kg (max 3506

mg/kg in leek). Low nitrate levels found in apple and higher levels in the

soft fruits. Levels in the frozen products were slightly below the fresh

products. Nitrite contents were very low in the fruit and vegetables (<1

mg/kg) [45].

The results of analyses of 1994 Total Diet Study (TDS) samples or

nitrate and nitrite were reported. There was a comparison between the

dietary intake of nitrate and nitrite with Acceptable Daily Intakes (ADI).

Nitrate concentrations were highest in green vegetables (444 mg/Kg),

potatoes (136 mg/Kg), and other vegetables (182 mg/Kg), were these food

groups made the greatest contributions to dietary intake of nitrate with

39%, 37%, and 30%, respectively. Nitrite concentrations were low [42].

33

A study on Spanish and sorrel grown under nitrogen fertilization

determined their nitrate contents which were (1131-1459 and 62-835

mg/Kg for Spanish and sorrel, respectively). It was also demonstrated that

nitrate contents were not distributed in vegetative parts. Stalks, leaf

petioles, and veins contained higher nitrate content [53].

Another study on spinach and beet determined their nitrate-nitrite

levels in commercially and in home processed beets and spinach. Home

processed beet were found to have higher nitrate contents than

commercially processed beets, while home frozen spinach showed greater

nitrate levels than commercially frozen spinach, although the difference

was not significant. Length of storage of home processed beets and spinach

did not affect the nitrate or nitrite content [11].

The nitrate contents in some species of wild edible herbs and

vegetables common in Jordan were determined using the cadmium

reduction method. The nitrate level varied widely ranging from 29 mg/kg

the leaves of tetragonolbus (Tetragonolbus palaestinus) to 6743 mg/kg in

fenugreek (Trigonella stellata ). In general, the nitrate content in the stems

was higher than in the leaves which in turn were higher than that in the

roots and bulbs. The same species collected from irrigated farms were

generally of higher nitrate content than those collected from pasture, forest,

or non-irrigated farms [60].

34

Chapter Three

Materials and methods

35

Materials and Methods

3.1. Chemicals and reagents:

All chemicals used were of analytical grade.

Activated aluminum (Aluminum oxides): Riedel-de Haen (Aluminum

oxides, neutral), for column chromatography: 30 cm length, and 2 cm in

diameter.

Nitrate reagent: HI93728-0, for nitrate determination.

NAD reagent: N-(1-naphthyl) ethylenediamine.2HCl: SIGMA.

Sulfanilamide reagent: Sulfanilamide (P-Aminobenzenesulfonamide):

SIGMA.

Other reagents used were: NaNO2, and CH3COOH.

3.2. Instruments:

Nitrate meter: HANNA Instrument, HI 93728-0.

UVPC spectrophotometer device: Schimadzu, UV-1601PC, UV-Visible

spectrophotometer.

36

3.3. Sample collection: Five vegetable crops were collected for this

study: cucumber, tomato, cabbage, potato, and onion. These crops are

the most common cultivated crops in Tulkarm district at this season. In

addition both cucumber and tomato fruits are usually consumed fresh,

and grown above the ground, however, potato and onion represent the

crops that grown under ground and the edible portion is the stem. On

the other hand cabbage represents the leafy vegetables.

Seventy five samples of all these crops were collected, each sample

represent a farm.

All samples were collected from two basic regions: As-Sha’raweya

area (location no.1), which is a flat land, known as an agricultural land

usually cultivated with almost all vegetable crops, especially Ateel- Al

Deir area.

The second region is Thenabe (location no.2), which is a high area

cultivated with different crops.

In addition to these two regions, cucumber samples were also

collected from a third region known as Al-Kafryat (location no.3).

Cucumber fruits were divided into two groups, large samples (>100

mm), and small samples (<100 mm).

Samples were collected between January and July 2004.

37

From each farm one fruit sample was collected. Farms and samples

were chosen randomly (Table 6).

Table (6): Number of samples collected from the different locations for each vegetable type.

Number of samples collected from each location

Type As.Sha'raweya No.1

Thenabe No.2

Al-Kafreyat No.3

Tomato 8 6 None

Onion 6 7 None

Potato 8 8 None

Cabbage 7 6 None

Cucumber 5 4 10

38

3.4. Preparation of reagents and standard solution:

a. NED reagent: 0.2 g N-(1-naphthyl) ethylenediamine.2HCl were

dissolved in 150 ml 15% of (V/V) CH3COOH. Filtered when it is

necessary, and stored in glass-stoppered brown glass bottle.

b. Sulfanilamide reagent: 0.5 g sulfanilamide were dissolved in 150 ml

of 15% CH3COOH (V/V). Filtered if necessary and stored in glass-

stoppered brown glass bottle.

c. Sodium nitrite standard solution: (1) stock solution (1000 ppm

NaNO2): 1 g NaNO2 was dissolved in H2O and diluted to 1L.

(2) Intermediate solution (100 ppm NaNO2): 100 ml of the stock

solution were diluted to 1L with H2O. (3) Working solution (1 ppm

NaNO2): 10 ml of the intermediate solution were diluted to 1L with

H2O.

3.5. Nitrate and nitrite extraction.

A fifty gram sample of the prepared crop was blended with 50ml

distilled water in a home blender. The mixture was filtered through

Whatman no.2 filter paper, and the filtrate was passed through a glass

39

column fitted with a tape and filled with Activated alumina, in order to

separate the green color (Chlorophyll) and get a transparent solution.

Water was used as eluting solvent. The eluted solution filtered using

0.45um filter paper in order to eliminate the turbidity and get a clear

solution.

3.6. Quantitative determination:

3.6.1. Quantitative determination of nitrate: 10ml of the

transparent, clear solution was analyzed for the nitrate content using

“HANNA Instrument” which gives the sample content of nitrate as

NO3-N. The concentration of NO3 can be calculated by multiplying the

nitrogen concentration by a factor of 4.43 to have the exact nitrate

(NO3) concentration in ppm of the sample.

3.6.2. Quantitative determination of nitrite: with the AOAC Official

Method 973.31. A portion of solution containing nitrite was

transferred into a 25 ml volumetric flask. Then 2.5 ml sulfanilamide

were added, followed by addition of 2.5 ml NAD. The volume was

completed with water and left for 15 minutes in order to give time for

color development. The absorbance was measured at 545 nm against a

blank solution. The nitrite concentration was determined using the

40

calibration curve prepared as follows: 10, 20, 30, and 40 ml of nitrite

working standard solution were transferred into 50 ml volumetric

flasks in order to prepare standard solutions of 0.2, 0.4, 0.6, and 0.8

ppm NaNO2. Then 2.5 ml of sulfanilamide reagent were added to each

flask followed by 2.5 ml NAD reagent. The volumes were completed

to the marks. The absorbances were measured after 15 minutes at 545

nm. The calibration curve was constructed by plotting the absorbance

vs. the concentration.

3.7. Statistical analysis: Data were analyzed using SPSS package. One

way ANOVA was conducted followed by a mean separation by LSD test.

41

Chapter Four

Results

42

Results

Nitrate and nitrite content in five vegetable crops were determined in

three areas of Tulkarm district as shown in Table (7) for nitrate, and in

Table (8) for nitrite.

It can be seen from Tables 7 and 8 that tomato fruits which were

collected at different stages of growth, showed the lowest average of

nitrate contents among other crops with an average of 17.95 and 15.96

mg/Kg, with a range of 0 - 34.12, and 0 - 58.14 mg/kg in locations 1 and 2,

respectively. While the average nitrite content in tomato fruits were 0.13

and 0.16 mg/Kg, with a range of 0.01 - 0.37 and 0.04 - 0.28 mg/Kg for

locations 1 and 2, respectively.

While the potato showed the highest levels of nitrate content,

average of 231.84 mg/kg, with a range of 90.9 - 526.28 in location 1, and

an average 274.42 mg/kg, with a range of 33.49 -562.28 mg/kg in location

2. While the average nitrite levels in potato were 0.54 and 0.84 mg/Kg,

with a range of 0.09 - 1.29 and 0 - 1.83 mg/Kg for locations 1 and 2,

respectively.

The dry onion results did not show high levels of nitrates. The

average nitrate content were 49.79, and 49.88 mg/kg, with a range of 18.43

- 66.34, and 2.76 - 105.97 mg/kg for regions 1and 2, respectively. The

43

average nitrite content in dry onion was 0.32 and 0.5 mg/Kg, with a range

of 0.07 - 0.78 and 0.04 - 1.46 mg/Kg in locations 1 and 2, respectively.

The average nitrate levels in cabbage were 85.23, and 198.46 mg/kg,

with a range of 0 - 306.2, and 115.68 - 273.88 mg/kg for locations 1 and 2

respectively. While the nitrite averages were 0.55 and 0.7 mg/Kg, with a

range of 0 - 2.98 and 0.16 - 1.78 mg/Kg in locations 1 and 2, respectively.

Table (7): Average nitrate (NO3) content in the vegetables surveyed in Tulkarm district.

Crops location

No. of samples

Mean (NO3) mg/kg ± S.E

Range (mg/Kg)

1 8 17.95 ± 3.76 0 - 34.12 Tomato

2 6 15.96 ± 9.28 0 - 58.14

1 6 49.79 ± 6.99 18.43 - 66.34 Onion

2 7 49.88 ± 13.77 2.76 - 105.97

1 8 231.84 ± 54.56 90.9 - 526.28 Potato

2 8 274.42 ± 79.21 33.49 - 562.28

1 7 85.23 ± 39.49 0 - 306.2 Cabbage

2 6 198.46 ± 25.52 115.68 - 273.88

1 10 95.20 ± 17.11 26.78 - 124.4

2 8 119.54 ± 18.79 53.56 - 235.84

cucumber

3 20 176.35 ± 19.71 44.05 - 328.26

*S.E: Standard Error of the mean.

44

Table(8): Average nitrite (NO2) content in the vegetables surveyed in Tulkarm district.

Crops Location No. of samples

Mean (NO2) mg/kg ± S.E*

Range (mg/Kg)

1 8 0.11 ± 0.05 0.01 - 0.37 Tomato

2 6 0.16 ± 0.04 0.04 - 0.28

1 6 0.32 ± 0.11 0.07 - 0.78 Onion

2 7 0.51 ± 0.18 0.04 - 1.46

1 8 0.54 ± 0.16 0.09 - 1.29 Potato

2 8 0.84 ± 0.27 0 - 1.83

1 7 0.55 ± 0.41 0 - 2.98 Cabbage

2 6 0.7 ± 0.25 0.16 - 1.78

1 10 1.78 ± 0.23 0.45 - 2.22

2 8 1.47 ± 0.83 0.17 -5.45

Cucumber

3 20 3.55 ± 0.69 0.15 - 8.05


45

The cucumber fruit samples were collected at different growth

stages from three different locations at Tulkarm district. The samples were

divided into two groups, in the first one the nitrate and nitrite content were

determined in small and large size cucumber fruits. In the second group the

nitrate and nitrite content were determined in the peel of the fruits and in

the extract of the cucumber fruits.

Table (9) shows the distribution of nitrate contents between large

size and small size cucumber samples.

Obtained results shows a higher nitrate levels in the small fruits than

in the large ones. Where the average levels of nitrate in the large size

cucumber were 53.03, 140.82, and 110.23 mg/kg, with a range of 24.19 -

82.93, 77.75 - 205.60, and 3.45 - 245.33 mg/Kg, from locations one, two,

and three, respectively, while the average nitrate levels in small size

cucumber were 83.12, 161.97, and 213.54 mg/kg, with a range of 29.37 -

165.86, 29.37 - 266.07, and 84.66 - 411.19 mg/Kg from locations 1, 2, and

3, respectively.

Table (10) shows the distribution of nitrite content in small size and

large size cucumber, where the nitrite levels in large size samples were

1.13, 0.45, and 1.58 mg/kg, with a range of 0.54 - 1.48, 0.03 -1.37, and

0.02 - 4.80 mg/Kg from locations one, two, and three, respectively. The

average nitrite levels in small size samples were 1.77, 2.7, and 4.48

46

mg/Kg, with a range of 0.37 - 2.95, 0.30 - 9.52, and 0.27 - 11.29 mg/Kg

from locations 1, 2, and 3, respectively.

Table (9): Average nitrate (NO3) levels in large size cucumber fruits.

Large size cucumber (>100mm)

Location

No

No.of

samples

Average NO3

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 53.03 ± 9.53 24.19 - 82.93

2 4 140.82 ± 27.23 77.75 - 205.6

3 10 110.23 ± 25.88 3.45 - 245.33


Table (10): Average nitrate (NO3) levels in small size cucumber fruits.

Small size cucumber (<100 mm)

Location

No

No.of

samples

Average NO3

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 83.12 ± 24.53 29.37 - 165.86

2 4 161.97 ± 51.41 29.37 - 266.07

3 10 213.54 ± 25.88 84.66 - 411.19


47

Table (11): Average nitrite (NO2) levels in large size cucumber fruits.

Large size cucumber (>100 mm)

Location

No

No.of

samples

Average NO2

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 1.13 ± 0.16 0.54 - 1.48

2 4 0.45 ± 0.32 0.03 - 1.37

3 10 1.58 ± 0.56 0.02 - 4.8


Table (12): Average nitrite (NO2) levels in small size cucumber fruits.

Small size cucumber (<100 mm)

Location

No

No.of

samples Average NO2

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 1.77 ± 0.50 0.37 - 2.95

2 4 2.7 ± 2.27 0.3 - 9.52

3 10 4.48 ± 0.56 0.27 - 11.29


48

Table (11) shows the distribution of the nitrate levels between the

peel and the essences of cucumber. The results shows that the peel of the

cucumber contains higher nitrate contents than the essence, where the

average nitrate levels in the peel were 140.98, 104.52, and 205.6 mg/kg

with a range of 91.57 - 236.69, 0 - 183.13, and 41.46 - 570.14 mg/Kg for

locations one, two, and three, respectively, while the average of nitrate

levels in the essence were 103.68, 70.835, and 176.05 mg/kg, with a range

of 0 - 292.06, 24.19 - 114.03, and 81.20 - 502.76 mg/Kg for locations 1, 2,

and 3, respectively.

Table (12) shows the distribution in nitrite levels in the peel and the

essences of cucumber. The average nitrite content in the essences are 2.29,

0.14, and4.25 mg/Kg, with a range of 1.42 - 3.82, 0.08 - 0.21, and 0.07 -

21.12 mg/Kg for locations one, two, and three, respectively, while the

average nitrite content in the peel were 1.91, 2.58, and 3.89 mg/Kg, with a

range of 0.78 - 3.80, 0 - 10.25, and 0.26 - 10.04 mg/Kg for locations 1, 2,

and 3, respectively.

49

Table (13): Average nitrate (NO3) content in (mg/Kg) in the essence of cucumber fruits.

Nitrate in essences

Location

No No. of

samples Average NO3

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 103.68 ± 49.57 0 - 292.06

2 4 70.84 ± 20.22 24.19 - 114.03

3 10 176.05 ± 38.79 81.2 - 502.76


Table (14): Average nitrate (NO3) content in (mg/Kg) in the peel of cucumber fruits.

Nitrate in peels

Location

No

No. of samples Average NO3

(mg/Kg)±S.E*

Range

(mg/Kg)

1 5 140.98 ± 37.29

91.57 - 236.69

2 4 104.52 ± 40.65

0 - 183.13

3 10 205.6 ± 52.35 41.46 - 570.14


50

Table (15): Average nitrite (NO2) content in (mg/kg) in the essence of cucumber fruits.

Nitrite in essences

Location No No.of samples Average NO2

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 2.29 ± 0.42 1.42-3.82

2 4 0.14 ± 0.27 0.08-0.2

3 10 4.25 ± 1.96 0.07-6.57


Table (16): Average nitrite (NO2) content in (mg/kg) in the peel of cucumber fruits.

Nitrite in peels

Location No No.of samples Average NO2

(mg/Kg) ± S.E*

Range

(mg/Kg)

1 5 1.91 ± 0.61 0.78 - 3.8

2 4 2.58 ± 2.55 0 - 10.25

3 10 3.89 ± 1.15 0.26 - 10.04


51

Chapter Five

Discussion

52

Discussion

Many studies were conducted to measure the nitrate and nitrite

content in vegetables. The levels of the nitrate obtained in this study were

compared with previous studies (Table 13).

It is clear that the result of our study shows a variation in the nitrate

and nitrite levels in the different crop samples. The levels of nitrate in

tomato were low compared to the other crops. However, for cucumber the

level was normal, and for both potato and cabbage the level was high.

Even with high levels obtained, the nitrate levels reported in our study does

not exceed the pollution level (NO3-N>325mg/Kg) [65]. More recent

surveys have shown no correlation between nitrate levels and the incidence

of methemoglobenemia until nitrate levels exceed 443 mg/Kg [66].

There are many factors that affect the nitrate level in crops, among

these factors are the agricultural system and practices, for instance it was

found that the nitrate level in vegetables grown under organic systems is

higher than that grown under conventional system [39].

Another important factor that interferes with the nitrogen

accumulation in plant tissues is the environmental and microclimate

conditions. It was shown that nitrate level is affected by the high sunlight

intensity [54], which increases the nitrate levels in plant tissues by

53

increasing the nitrate reductase activity which converts the nitrogen in

plant to nitrate.

Growing conditions like temperature, stress (drought), hot or dry

winds affect the nitrate accumulation in plants [WHO,1995] [5, 15], it has

been found that vegetables grown in winter, the time of year with low

temperature and less sunlight, has a higher nitrate content [European

Commission,1998] [27, 54].

54

Table (17): Comparison of nitrate (NO3) content in the vegetables surveyed in Tulkarm district, and the nitrate content reported in other studies.

Nitrate content in the present work

Reported nitrate content in other studies

Crops Nitrate content (mg/kg)

Nitrate content (mg/Kg)

Reference

5.3-144.3 Markowska.A,et al.(1995)

4.43-119.61 T.M.Knight et al.(1987)

Tomato 16.955

20.8-191.9 Agopov et al.(1990)

0-1.9 Amr and Hadidi (2001) Onion 49.835

48 MAFF no.158 (1998)

118-273 Leszezynska (1996)

219-350 MAFF no.91 (1996)

75-283 MAFF UK no.163 (1998)

Potato 253.13

136 MAFF UK no.137 (1994)

460.7-910 Knight et al (1987)

338 MAFF no.158 (1998)

Cabbage 141.845

165-351 MAFF no.91 (1996)

Cucumber

122.72 46.95-138.78 Amr.2000

*MAFF: Ministry of Agriculture, Fisheries & Food.

55

Table (18): Comparison of nitrite (NO2) content in the vegetables surveyed in Tulkarm district, and the nitrate content reported in other studies.

Nitrite content in the present work

Reported nitrite content in other studies

Crops

Nitrite content (mg/kg)

Nitrite content (mg/Kg)

Reference

Tomato 0.1475 0-0.31 Markowska et al.(1995)

0.44 Knight et al. (1987)

Potato 0.69 ND-0.8 MAFF no. (163) (1997) and no.(137) (1994)

Cabbage 0.625 0.5 Knight el. al. (87)

Cucumber 2.02 0.78 Amr.2000

*ND: Not Detected.

Therefore, vegetables grown in heated greenhouses have higher

nitrate contents than those grown in open fields or tunnels , because of the

lower light intensity, and higher nitrogen mineralization [59, 41, 7].

The type of crop is also among the primary factors that affect the

nitrate level [13]. Foods that tend to accumulate large amount of nitrate

include leafy vegetables such as spinach, lettuce, and cabbage, and root

vegetables such as carrots, beet, and broccoli, though at a much lower level

than do the leafy vegetables [61].

56

The herbicide application may also affect the nitrate accumulation in plants

[15, 63].

Other factors that may affect the nitrate concentrations in

vegetables: shading rate which increased the nitrate content [72], and

harvesting time [52], where late harvested vegetables had the lowest nitrate

levels, although, the harvest date had no significant effect on either nitrate

or nitrite content of the greenhouse grown vegetables [8].

All these factors as well as other factors related to local

environmental conditions might be the reason behind the variation in the

results we obtained with that of other results

The tomato fruits show the lowest nitrate content among all the

other crops tested, the same results were found in all other studies

conducted previously. This might be related to the types of the segments

present and the lack of chlorophyll in the tomato crops. This may explains

the high nitrate content in green vegetables and cucumber which shows a

higher nitrate content than tomato.

Higher nitrate content was detected in small size cucumber. That

might be due to the stage of growth. During initial growth, the nitrate taken

up by the plant is used for root and shoots development. At this stage, the

roots are able to take up more nitrate than required and it accumulates in

57

the stems and leaves of the plants. As the plants develops, the leaves of the

plant are able to convert more nitrate into plant protein, therefore less

nitrate is found in the plant as it matures.[15, 63]

It was also reported by [12] that the NRA (Nitrate Reductase

Activity) was greater in leaves (higher in young leaves). The NRA in leaf

and root increased with increasing the nitrate supply.

Comparing the nitrate content in the essence and the peel of the

cucumber fruits shows a higher nitrate content in the peel which may be

due to the presence of the chlorophyll.

In a study on cucumber, it was reported that the highest

concentrations of nitrate were found in the neck, and skin tissues of

cucumber fruits.[31]

There are other factors that could affect the nitrate content in

cucumber fruits such as the nutrient medium. The concentration of nitrate

in all regions of the cucumber fruit was higher when nitrate constituted

75% or more of the total nitrogen in the nutrient medium, but was reduced

by increasing the concentrations of the ammonium (NH4) [31].

Comparison of nitrate content in underground stem samples (onion

and potato) shows a lower nitrate content in onion, and this might be due to

the fertilization program. Most of the farmers do not apply a specific

58

fertilization program; they usually plant onion beside the greenhouse as a

complementary crop.

Potato shows a high nitrate content; this is consistent with the

previous studies that show high concentrations of nitrate, this might be due

to the high application of nitrogen fertilization during the growing season.

The late application of nitrogen fertilizers results in higher accumulation in

both leaves and potato tuber [20].

In a study, it was reported that lettuce and potato consumption make

the greatest contribution to dietary intake of both nitrate and nitrite [59].

Cabbage shows high nitrate content in comparison with other

studied and reported results.

The nitrate levels in leafy vegetables are higher than bulb, root,

shoot, inflorescence, and tuber vegetables [58] [41].

Green leafy and root vegetables tend to accumulate large amount of nitrate

[62] [61].

The nitrate and nitrite content in several crops collected from two

different regions in Tulkarm district (Al-Sha'raweya and Thenabe) were

analyzed to determine if the location has an effect on the nitrate and nitrite

59

concentration in crops. The results show no significant effect of the region

on the nitrate and nitrite content in different vegetable crops.

The nitrite levels generally were below the allowable range. The

nitrite content must be lower than 1mg/Kg [13] [59]. The nitrite content

results in this survey was lower than 1mg/Kg except in some cucumber

samples which were higher than 1mg/Kg, but still in an acceptable range

and propose no danger on human health. It was reported that the nitrite

levels start to be dangerous if it is higher than 100 mg/Kg [54] which is not

the case here.

60

Chapter Six

Conclusions

61

6. Conclusions and Recommendations

In spite of the miss use of fertilizer practiced by the farmers, the

accumulation of both nitrate and nitrite content in samples of different

cultivated crops shows that tomato fruits contain the lowest nitrate content,

while potato and cabbage shows a relatively high nitrate levels, onion did

not show high nitrate levels.

The study indicated that the size of fruit in cucumber has an effect

on its nitrate content; large fruit contains less nitrate content.

In addition the result proved that the cucumber fruit peel contains

high nitrate content than the pulp. The result also shows a high nitrate

content in cabbage than other crops. Similar findings were shown with

potato tuber samples.

The nitrite levels were below 1mg/Kg which is below the permitted

limit of nitrite levels in vegetables, which agreed with all other previous

studies.

The nitrate and nitrite levels in vegetables cultivated in Tulkarm

district are below the pollution level number four, and does not pause the

consumers to health risks.

62

In accordance of the results we obtained, we highly recommend that

more work to be conducted on this subject. Both governmental and

nongovernmental organizations have to focus on this issue and must

consider it as part of their strategy. The education and research centers

have to perform more comprehensive survey on other agricultural crops

and on different production areas. The aim of this work was mainly to

highlight this important and crucial issue which has a direct effect on our

health and environment.

63

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70

Appendices:

Appendix (a): The area (dunum) cultivated with vegetable croups in Tulkarm district in (2003-2004).

Protected vegetables Unprotected vegetables Cluster

tomato cucumber

others

tomato cucumber

squash

Qafeen 7 55 0 2 0 0

Nazlet-essa

1 43 1.5 4 0 0

Al-Nazla Al-Sharqeya

1 26 0 7 0 5

Baqa As-Sharqeya

15 190 0 10 0 43

Al-Nzla Al-Wosta

0 0 0 0 0 3

Nazlat abu-Nar

0 0 0 0 0 5

Al-Nazla Al-Gharbeya

0 25 0 4 0 0

Zeata 30 196 0 12 0 18

Saida 1 55 0 8 0 41

Elar 18 116 0 0 0 38

Deir Alghoson

10 160 0 0 2 4

Bal'a 5 28 0 0 0 0

Thenaba 20 282 0 15 3 0

Tulkarm 20 253 0 16 12 43

Anabta 0 16 0 0 0 5

Rameen 0 2 0 0 0 7

71

Far'on 7 15 0 0 4 10

Shoofe 10 70 0 3 0 3

Kherbet Gbara

1 27 0 0 0 2

Beit Leed 0 0 0 0 0 8

Kofr Soor

0 2 0 0 0 0

Kofr Gammal

1 47 0 0 0 0

Kofor A'boush

20 0 0 0 0 0

A'teel 1 245 0 15 0 53

Kofr Al-Labad

168 21 0 0 0 0

Total 336 1874 1.5 96 21 288

*Ministry of agriculture in Tulkarm district

72

Appendix (b): The area (dunum) cultivated with unprotected vegetables and field crops in of Tulkarm district (2003-2004).

Unprotected vegetables Field crops Cluster

Leafy vegetables

Green legumes

tubers others cereals tubers Dry legumes

ornamental

Qafeen 21 25 5 7 280 5 16 0

Nazlet-essa 51 23 6 43 190 4 17 0

Al-Nazla Al-

Sharqeya

10 12 11 10 95 3 0 0

Baqa As-Sharqeya

131 7 28 150 60 6 0 0

Al-Nzla Al-Wosta

8 6 4 2 30 0 0 0

Nazlat abu-Nar

0 3 0 0 23 9 0 0

Al-Nazla Al-

Gharbeya

49 22 24 71 56 2 0 0

Zeata 25 55 39 31 294 20 21 0

Saida 47 53 41 128 78 35 10 0

Elar 184 32 27 280 271 66 0 0

Deir Alghoson

64 39 16 34 188 56 3 0

Bal'a 3 6 5 0 503 7 0 0

Kofr Roman

0 2 0 10 35 0 0 0

Thenaba 185 29 41 126 231 234 0 0

Tulkarm 140 141 138 86 1540 171 48 4

Anabta 2 26 6 7 455 50 7 3

Rameen 0 15 18 0 230 28 6 2

Far'on 20 14 2 15 271 6 0 0

Shoofe 25 22 0 0 165 22 0 0

Kherbet Gbara

0 0 0 5 140 0 0 0

Safareen 0 5 0 0 460 12 0 4

73

Beit Leed 0 23 22 0 340 6 9 2

Kofr Soor 0 0 0 0 50 0 1 4

Koor 0 0 0 0 130 0 0 0

Kofr Zeebad

0 0 0 0 190 0 5 3

Kofr Gammal

0 8 0 4 428 0 15 4

Kofor A'boush

0 0 0 0 500 0 11 5

A'teel 67 47 53 132 290 132 6 0

Kofr Al-Labad

0 11 4 0 620 0 0 0

Total 1032 628 490 1141 8143 874 175 31

*Ministry of agriculture in Tulkarm district

74

Appendix (c): The most cultivated trees and their areas (dunum) in Tulkarm district for years (2003-2004).

Cluster Olive Citrus Stone fruits

Almond Fig

Qafeen 8045 194.5 0 350 0

Nazlet-essa

993 129 0 50 0

Al-Nazla Al-

Sharqeya

800 53 0 40 4

Baqa As-Sharqeya

785 192 4 20 0

Al-Nzla Al-Wosta

190 0 0 60 _

Nazlat abu-Nar

405 0 0 40 0

Al-Nazla Al-

Gharbeya

95 74 0 50 0

Zeata 521 141 18 30 5

Saida 2570 0 1000 800 12

Elar 6570 369.5 400 2500 10

Deir Alghoson

9292 25.5 35 195 12

Bal'a 15100 0 1011 900 100

Kofr Roman

2200 36 0 50 5

Thenaba 402 49 5 30 3

Tulkarm 5741 795 22 285 24

Anabta 8930 22.5 5 650 35

Rameen 3408 0 55 250 75

Far'on 1634 294 5 30 0

Shoofe 5074 264 0 100 2

75

Kherbet Gbara

330 0 0 20 0

Safareen 2685 0 25 90 15

Beit Leed

10386 0 95 250 100

Kofr Soor

2565 0 5 80 2

Koor 1765 0 5 30 2

Kofr Zeebad

2850 0 5 40 2

Kofr Gammal

2800 259 6 120 4

Kofor A'boush

4497 0 2 60 0

A'teel 5259 103.4 3 150 5

Kofr Al-Labad

9293 0 4 250 4

Total 115185 3001.4 2710 7520 431

* Ministry of Agriculture in Tulkarm District.

In West Bank, the nitrate pollute a lot of the water sources. In Tulkarm the percentage of water not polluted with nitrate is 27%, while it is about 23% in Qalqelia. The average nitrate is higher than 50 mg/l in 14% of wells water (ground water) (55)

76

Appendix (d): Descriptive statistics of chemical and physical parameters of the wells studied in Tulkarm district [60].

Variable Minimum Maximum Average Std.Deviation

EC (MS/cm)

209.00 2155.00 910.30 299.30

TDS (mg/L)

272.00 1194.00 508.60 .171.60

pH 7.22 8.25 7.73 0.27

Ca+2 (mg/L)

54.30 161.50 95.10 20.40

Mg+2

(mg/L) 7.60 40.90 22.60 7.70

Na+1 (mg/L)

11.30 140.00 54.10 240.20

K+1 (mg/L) 0.80 35.00 5.80 7.40

HCO3-1

(mg/L) 130.20 526.80 308.40 69.90

SO4-2

(mg/L) 4.80 143.30 25.80 21.60

Cl-1 (mg/L) 29.40 419.80 86.50 66.90

NO3-1

(mg/L) 13.30 260.20 64.50 48.00

77

Appendix (e): Physical and chemical results obtained from the wells studied in Tulkarm district [60].

Name Location EC

MS/cm

pH Ca+2

mg/L

Mg+2

mg/L

Na+

mg/L

K+

mg/L

HCO3

-

mg/L SO4

-2

mg/L

Cl-

mg/L

NO3

-

mg/L

Said Jaber Shwayka 783 7.80

66.1 38.6 27.0 8.5 314.0 16.5 61.5 46.0

M.Makkawi 'Allar 735 7.80

90.5 17.8 30.2 2.4 285.1 12.1 45.0 78.8

R.Samara 'Allar 1024 7.61

108.6

19.8 72.1 2.2 326.0 28.0 116.2

69.7

A.M.Qasem "Attil 976 7.49

100.4

22.7 65.1 2.1 370.0 22.5 74.5 76.5

H.Amous 'Attil 965 7.37

120.1

16.8 48.8 5.9 296.3 19.1 86.2 127.0

J.Awartani Anabta 1335 7.83

127.5

31.4 100.0

2.4 403.4 28.0 151.0

104.0

Municipality well1

Anabta 1063 7.23

109.4

23.7 61.0 22.1 238.0 39.1 97.0 205.7

N.Mousa Anabta 696 7.54

88.9 13.8 30.2 2.8 269.0 19.3 59.8 32.6

Bal'a Bal'a 594 8.00

56.9 24.3 24.0 2.4 266.5 6.8 38.8 13.3

S.Said Baqa A-Sharqiyya

751 7.54

98.7 14.8 30.2 2.3 273.9 15.5 39.2 102.8

Municipality well

Deir Al Ghsun

706 7.62

87.2 17.8 27.9 1.3 200.8 13.3 50.9 125.6

Far'on Far'on 634 7.55

75.7 23.2 13.6 2.4 275.2 10.7 38.2 35.3

I.Attir Far'on 631 7.53

72.4 26.2 11.9 0.8 221.9 39.3 38.2 51.1

78

A.A.Shanab Irtah 1070 7.86

112.3

32.2 43.4 22.0 430.1 23.6 78.1 52.6

R.Qubbaj Nur Shams

1139 7.75

85.3 30.9 105.0

0.9 379.3 19.0 142.0

43.0

A.R.Mir'eb Shwayka 892 7.98

104.7

23.1 38.6 4.4 371.8 15.4 64.4 42.0

Said Jaber Shwayka 721 7.92

76.5 24.2 30.2 3.4 250.4 14.5 59.8 67.6

Asa'd Taffal

Thennaba

614 8.12

70.7 15.0 28.9 4.9 258.8 13.0 43.0 27.3

A.Q Quzmar

Tulkarm 989 8.03

97.2 20.0 65.1 22.0 347.7 36.6 93.7 51.6

Municipality well2

Tulkarm 1298 8.01

111.9

39.7 90.0 8.5 417.8 38.0 113.6

98.0

M.Said Tulkarm 2155 8.10

161.5

36.4 240.0

2.4 526.8 96.0 359.7

26.0

A.J.Samara Zeita 731 7.52

86.4 17.3 34.9 2.2 300.0 18.1 54.9 30.6

80

.

.

(NO3) (NO3)

.75

)(

)(.

(NO3) HANNA instrument (HI93728-0)

(NO2)AOCA Official Method

973.31.

(NO3)(NO3)

(NO3)253.13/

81

(NO3)16.95/.

(NO3)

(NO3).

(NO3)

).(

(NO2))

1/(.

(NO3)(NO2).

.

(NO3)(NO2)

.

.